Vitamin D3 Flourinated Analogs

ABSTRACT

Compounds of the formula    &lt;IMAGE&gt; I  wherein R is hydrogen, hydroxy, or fluorine, and X is H2 are useful as agents for the treatment of hyperproliferative disorders of the skin such as psoriasis, as agents for the treatment of cancer, such as, leukemia, and as agents for the treatment of sebaceous gland diseases, such as, acne and seborrheic dermatitis.

This is a continuation of application Ser. No. 07/971,788 filed Nov. 5,1992, now abandoned which is a continuation-in-part of Ser. No.07/957,500, filed Oct. 7, 1992.

BRIEF SUMMARY OF THE INVENTION

The invention relates to compounds of the formula ##STR2## wherein R ishydrogen, hydroxy or fluorine and X is H₂ or ═CH₂.

Compounds of formula I as described above are useful as agents for thetreatment of hyperproliferative skin diseases such as psoriasis.Compounds of formula I as described above are also useful as agents forthe treatment and prevention of leukemia and cancer. Compounds offormula I above are also useful as agents for the treatment of sebaceousgland diseases, such as, acne and seborrheic dermatitis.

DETAILED DESCRIPTION OF THE INVENTION

In the formulas presented herein, the various substituents areillustrated as joined to the nucleus by one of the following notations:a wedged solid line indicating a substituent which is above the plane ofthe molecule, and a wedged dotted line indicating a substituent which isbelow the plane of the molecule.

The invention relates to compounds of the formula ##STR3##

wherein R is hydrogen, hydroxy or fluorine and X is H₂ or ═CH₂.

Compounds of formula I as described above stimulate differentiation anddecrease proliferation of human keratinocytes. Accordingly, compounds offormula I as described above are useful as agents in the treatment ofhyperproliferative skin disorders such as psoriasis, basal cellcarcinomas, disorders of keratinization, and keratosis. The compounds offormula I are also useful as agents in the prevention and treatment ofleukemia and cancer. The compounds of formula I are also useful for thetreatment of sebaceous gland diseases, such as, acne and 0 seborrheicdermatitis.

The invention also relates to a composition comprising a compound offormula I, or a mixture of two or more compounds of formula I. ]5

The invention also relates to a method for treating the above-mentioneddisease states by administration of a compound of formula I, or amixture of two or more compounds of formula I.

The invention also relates to a process for preparing compounds offormula I as described above.

Exemplary compounds of formula I of the invention are:

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23yne-19-nor-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol;and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

In the compound of formula I, R is preferably hydroxy or fluorine.

Preferred compounds of formula I are:

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;and

1,25-dihydroxy-16-ene-23-yne-26,26,26,27,27,27-hexafluoro-19-norcholecalciferol.

The compounds of formula I are prepared as hereafter described, withparticular reference to the Formula Schemes below. ##STR4##

In above Formula Scheme I, the compound of formula II, a known compound,is converted to the compound of formula III by reaction with a base,such as, n-butyllithium, and hexafluoroacetone. The reaction isconducted in an ether solvent such as tetrahydrofuran at about -50° C.to about -100° C. The compound of formula III is recovered by quenchingthe reaction, followed by a conventional work-up and a purification asby chromatography. ##STR5##

In reaction Scheme II, the .compound of formula III is deprotected togive the compound of formula IV by reaction with tetrabutylammoniumfluoride in an ether solvent such as, tetrahydrofuran.

The compound of formula IV is reacted with pyridinium chlorochromate ina chlorinated hydrocarbon solvent such as methylene chloride at roomtemperature to give the compound of formula V. ##STR6##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula scheme III, the compound of formula V is reacted withn-butyllithium and the compound of formula VI in a mixture of hexane andtetrahydrofuran at a temperature of -75° C. to give a compound offormula Ia after removal of silyl protecting groups withtetrabutylammonium fluoride in tetrahydrofuran solvent. ##STR7##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula Scheme IV, the compound of formula V is reacted withn-butyllithium and the compound of formula VII in a mixture of hexaneand tetrahydrofuran, at a temperature of -75° C. to give the compound offormula Ib after removal of the silyl protecting group withtetrabutylammonium fluoride in tetrahydrofuran solvent. ##STR8##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula Scheme V, the compound of formula V is reacted withn-butyllithium and the compound of formula VIII in a mixture of hexaneand tetrahydrofuran solvent at a temperature of -75° C. to give thecompound of formula Ic, after removal of the silyl protecting group withtetrabutylammonium fluoride in tetrahydrofuran solvent. ##STR9##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula Scheme VI, the compound of formula V is reacted withn-butyllithium and the compound of formula IX, a known compound, in amixture of hexane and tetrahydrofuran solvent at a temperature of -75°C. to give the compound of formula Id, after removal of the silylprotecting group with tetrabutylammonium fluoride in tetrahydrofuransolvent. ##STR10##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula Scheme VII, the compound of formula V is reacted withn-butyllithium and the compound of formula X, which can be prepared byknown methods, in a mixture of hexane and tetrahydrofuran solvent at atemperature of -75° C. to give the compound of formula Ie, after removalof the silyl protecting group with tetrabutylammonium fluoride intetrahydrofuran solvent. ##STR11##

wherein t.Bu is tertiarybutyl and Ph is phenyl.

In formula Scheme VIII, the compound of formula V is reacted withn-butyllithium and the compound of formula XI, which can be prepared byknown methods, in a mixture of hexane and tetrahydrofuran solvent at atemperature of -75° C. to give the compound of formula If , afterremoval of the silyl protecting group with tetrabutylammonium fluoridein tetrahydrofuran solvent.

The compounds of formula I as described above can be administeredorally, for the treatment of neoplastic diseases such as leukemia, towarmblooded animals which need such treatment. More specifically, thecompounds of formula I as described above can be administered orally toan adult human in dosages that are in the range of about 0.1 to 10 μgper day for the treatment of neoplastic diseases such as leukemia.

The compounds of formula I as described above, can be administeredorally in an effective amount, for the treatment of hyperproliferativeskin diseases such as psoriasis, basal cell carcinomas, disorders ofkeratinization, and keratosis, to warmblooded animals which need suchtreatment. Preferably, the compounds of formula I as described above canbe administered orally to an adult human in dosages that are in therange of about 0.001 to 100 μg per day for the treatment ofhyperproliferative skin diseases such as psoriasis, basal cellcarcinomas, disorders of keratinization, and keratosis.

The compounds of formula I as described above, can be administeredorally in an effective amount, for the treatment of sebaceous glanddiseases such as acne and seborrheic dermatitis to a host requiring suchtreatment. Preferably, the compounds of formula I, as described above,can be administered orally to an adult human in dosages that are in therange of about 0.07 μg to 770 μg per day, more preferably in the rangeof about 0.7 μg to 70 μg per day for the treatment of sebaceous glanddiseases, such as acne.

The compounds of formula I, as described above, can be administeredtopically in an effective amount, for the treatment ofhyperproliferative skin diseases such as psoriasis, basal cellcarcinomas, disorders of keratinization, and keratosis, to warmbloodedanimals which need such treatment. Preferably, the compounds of formulaI as described above can be administered topically in dosages that arein the range of about 0.01 to about 100 μg per gram of topicalformulation per day, for the treatment of hyperproliferative skindiseases such as psoriasis, basal cell carcinomas, disorders ofkeratinization, and keratosis.

The compounds of formula I, as described above, can be administeredtopically in an effective amount, for the treatment of sebaceous glanddiseases, such as acne and seborrheic dermatitis, to a host in need ofsuch treatment. Preferably, the compounds of formula I, as describedabove, can be administered topically in dosages that are in the range ofabout 0.1 to about 1000 μg per gram of topical formulation per day, forthe treatment of sebaceous gland diseases, such as, acne and seborrheicdermatitis.

The useful activity of compounds of formula I as agents for thetreatment of hyperproliferative skin diseases can be demonstrated by thefollowing test procedures which are known in the art, and which are alsoset forth in Holick et al., The Society for Investigative Dermatology,p. 709-714 (1986).

Human Keratinocyte Antiproliferative Assay

Cells: Primary or passage 1 subconfluent cultures of human neonatalkeratinocytes were grown in Keratinocyte Growth Media® (KGM® modifiedMCDB 153, Clonetics, Inc. Catalog #CC3001) supplemented with antibioticsor calcium chloride as needed. Cultures were obtained from neonatalforeskin epithelial keratinocytes using standard procedures.

Culture Conditions: Human neonatal foreskins were collected bycircumcision and placed into tubes containing Dulbecco's minimumessential Media (DMEM) with 10% serum. Upon arrival at the laboratory,they were mechanically trimmed of excess dermis, and treated with asolution of trypsin/ethylenediamine tetraacetic acid (EDTA)(0.05%/0.02%) at 4° C. overnight. The epidermis was stripped from thedermis, agitated in buffered saline to remove basal keratinocytes andthe stratum corneum later removed. The separated cells were centrifuged,resuspended in media, counted, and the cells plated onto plastic culturedishes or flasks at 5,000 to 25,000 cells/cm² in KGM media according toprotocols developed by Boyce and Ham, In Vitro Models for CancerResearch III, 246-274, (1986) for MCDB 153 media. The cultures areincubated in humidified chambers with 5% CO₂ at 37° C. with refeedingfresh media 2 to 3 times per week. Prior to reaching confluency, thecells are replated (called passage 1) at 25,000 cells/well on 6-wellcluster plates (Costar catalog #3406) in KGM.

Antiproliferation Assay Protocol: Approximately twenty-four hours afterpassage, the cells were refed with fresh KGM media supplemented to 1.5mM CaCl₂ that contains test compound or vehicle. Solutions of testcompounds were prepared as follows: 1 milligram quantities were receivedin amber glass vials and stored at -20° C. Sufficient 100% ethanol wasadded directly to vials to obtain a millimolar solution that wassubsequently aliquoted into small amber vials, overlayed with argon gasand stored at -20° C. Each stock solution was thawed once, used anddiscarded. Stock solutions were used within 4 to 6 weeks. Aliquots fromthe stock solution were diluted directly into medium and then seriallydiluted from micromolar to picomolar concentrations. Compounds weretypically tested at four concentrations in triplicate wells. Controlwells are supplemented with vehicle alone at the highest concentrationsuch as 0.1% ethanol. At the termination of the experiment prior to thecultures reaching confluency, the cells were enumerated by the followingprocedure. Dishes were washed with phosphate buffered saline, and thenincubated with trypsin/EDTA solution for 30 minutes. Cells weresuspended and an aliquot placed into isotonic buffered saline andcounted on an electronic particle counter (Coulter Counter). The counterwas periodically calibrated for the correct size of keratinocytes. Eachwell was counted in triplicate. The number of cell/dish was calculatedaccording to dilution factors used and results are presented as percentinhibition from cell numbers obtained in control cultures. The resultsare set forth in Table I.

                                      TABLE I                                     __________________________________________________________________________                 % Inhibition                                                                  Dose (M)                                                                      10.sup.-9                                                                            10.sup.-8                                                                            10.sup.-7                                                                            10.sup.-6                                   __________________________________________________________________________    1,25-dihydroxy-16-                                                                         99.6 ± 4.6                                                                        70.5 ± 8.5                                                                        45.6 ± 8.4                                                                        33.7 ± 8.8                               one-23-yne-26,27-                                                             hexafluorocholecalciferol                                                     25-hydroxy-16-ene-                                                                         119.2 ± 13.6                                                                      105.7 ± 12.1                                                                      75.7 ± 11.3                                                                       19.7 ± 5.3                               23-yne-26,27-                                                                 hexafluorocholecalciferol                                                     1α-fluoro-25-hydroxy-                                                                95.5 ± 1.5                                                                        96.6 ± 1.7                                                                        53.4 ± 3.32                                                                       25.8 ± 3.6                               16-ene-23-yne-26,27-                                                          hexafluorocholecalciferol                                                     __________________________________________________________________________

1,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol exhibitedan average ED₅₀ (Dose that would obtain 50% of the number of cells ascompared to the control)=0.08 μM.

25-hydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol exhibited anaverage ED₅₀ =0.5 μM.

1α-fluoro-25-hydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferolexhibited an average ED₅₀ =0.1 μM.

The useful activity of compounds of formula I as agents for thetreatment of neoplastic diseases, such as leukemia, can be demonstratedby the following test procedures.

HL-60 Differentiation Assay:

The HL-60 tumor cell line was originally derived from a patient withpromyleocytic leukemia and purchased from American Type CultureCollection (ATCC CCL240). The cells are maintained in suspension usingthe media RPMI 1640 (Gibco catalog # 320-1875) plus 20% heat inactivatedfetal bovine serum (FBS). For experiments, cells are diluted to 200,000cells/ml in 5 ml of media in 25 cm² flasks. Compounds were typicallytested at four concentrations in duplicate flasks. Control flasks weresupplemented with vehicle alone at the highest concentration such as0.1% ethanol. Flasks are incubated upright for 4 days in 5% CO₂ at 37°C. On day 4, a 1 ml aliquot of cells was centrifuged, the media removedand the cells resuspended in a 0.2 ml of solution of nitrobluetetrazolium/phorbol 12 myristate 13-acetate (NBT/TPA) in media preparedas follows. Nitroblue tetrazolium is dissolved in media at 1 mg/ml. Tothis solution is added TPA to a final concentration of 100 ng/ml. Thecells are suspended and incubated at 37° C. for 30 min. prior totransferring to ice. An aliquot is removed and a total of 200 cells iscounted using a hemocytometer. Cells without pigmented granules arejudged to be undifferentiated while those containing blue black formazan(indicating conversion of NBT) granules were scored as differentiated.Results are expressed as percent differentiated cells by calculating theratio of the number of dark cells per total number of cells counted. Theresults are set forth below in Tables IIA and IIB.

                  TABLE IIA                                                       ______________________________________                                                     % Differentiated Cells                                                        10.sup.-10                                                                          10.sup.-9                                                                             10.sup.-8                                                                            10.sup.-7                                                                           10.sup.-6                             ______________________________________                                        1,25-dihydroxy-16-     64      89   91    90                                  ene-23-yne-26,27-                                                             hexafluorocholecalciferol                                                     25-hydroxy-16-ene-                                                                           NT      15      18   72    88                                  23-yne-26,27-                                                                 hexafluorocholecalciferol                                                     1α-fluoro-25-hydroxy-                                                                  NT      61      90   93    96                                  16-ene-23-yne-26,27-                                                          hexafluorocholecalciferol                                                     1,25-dihydroxy-16-                                                                           1.5     56.5    89.5 NT    NT                                  ene-23-yne-26,27-                                                             hexafluoro-19-nor-chole-                                                      calciferol                                                                    ______________________________________                                         NT means not tested.                                                     

1,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol exhibitedan average ED₅₀ (Dose that would induce at least 50% of cells todifferentiate)=<1 nM.

25-hydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol exhibited anaverage ED₅₀ =35 nM.

1α-fluoro-25-hydroxy-16-ene-23-yne-26, 27-hexafluorocholecalciferolexhibited an average ED₅₀ =<1 nM.

1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferolexhibited an average ED₅₀ =0.8 nM.

Additional tests were conducted on the following compounds for which theresults are provided in Table IIB.

                                      TABLE IIB                                   __________________________________________________________________________                % Differentiated Cells                                                        10.sup.-10                                                                        3 × 10.sup.-10                                                                10.sup.-9                                                                        3 × 10.sup.-9                                                                10.sup.-8                                                                        3 × 10.sup.-8                                                                10.sup.-7                                                                        10.sup.-6                            __________________________________________________________________________    1,25-dihydroxy-16-ene-                                                                    4   17    66.5                                                                             92.5 94.5                                                                             95.5 NT NT                                   23-yne-26,27-hexafluoro-                                                      cholecalciferol                                                               25-hydroxy-16-ene-                                                                        NT  NT    16 NT   43 NT   85 92                                   23-yne-26,27-hexafluoro-                                                      cholecalciferol                                                               __________________________________________________________________________     1,25-dihydroxy-16ene-23-yne-26,27-hexafluorocholecalciferol exhibited an      ED.sub.50 = 0.6 nM.                                                           25hydroxy-16-ene23-yne-26,27-hexafluorocholecalciferol exhibited an           ED.sub.50 = 25 nM.                                                       

The useful activity of compounds of formula I as agents for thetreatment of sebaceous gland diseases, such as acne and seborrheicdermatitis, can be demonstrated by the following test procedure.

Sebaceous cells are isolated from adult human sebaceous glands, derivedfrom facial skin removed during cosmetic surgery, and cultured on alayer of mouse 3T3 fibroblasts. This method involves the separation ofthe epidermal layer from the dermis by an electrokeratome. The dermaltissue is then treated, by enzymatic and mechanical methods, to generatea single cell suspension of sebaceous cells.

The cells are cultured in Iscove's medium containing 10% fetal calfserum and 4 μg/ml dexamethasone.

Cells are plated in medium without the test compound and then given thecompound in fresh medium 24-48 hours after the initial plating. Thecultures are given fresh medium, containing the test compound, every 48hours. On the day of harvesting, the cultures are rinsed with 0.03%ethylenediamine tetraacetic acid (EDTA) in phosphate buffered saline(PBS), to remove only the 3T3 fibroblasts. The remaining sebocytecolonies are incubated in 0.05% trypsin/0.03% EDTA to create a singlecell suspension of sebocytes. The cells are diluted, mixed vigorously tomaintain a single cell suspension, and counted in a hemocytometer.

All compounds are handled in the following manner. Stock solutions aremade up as 10⁻² M solutions in degassed 100% ethanol and stored at -20°C. in the dark. Solutions are never used after storage of more than amonth. During experimental use the solutions, which have been aliquoted,are thawed once and used by diluting directly into complete medium tothe appropriate concentration, at, 10⁻⁷, 10⁻⁸ and 10⁻⁹ M.

The compounds were tested for the inhibition of proliferation ofsebaceous cells in vitro at the following concentrations:

10⁻⁷, 10⁻⁸ and 10⁻⁹ M.

The results are summarized in Table III below as the amount of compoundnecessary to inhibit the proliferation of the sebaceous cells by 50% ascompared to a control, diluent treated only, culture (ID₅₀).

                  TABLE III                                                       ______________________________________                                        Compound           ID.sub.50 (μM)                                          ______________________________________                                        1,25-dihydroxy-16- <0.01                                                      ene-23-yne-26,27-                                                             hexafluorocholecalciferol                                                     25-hydroxy-16-ene- 0.05                                                       23-yne-26,27-                                                                 hexafluorocholecalciferol                                                     1α-fluoro-25-hydroxy-16-                                                                   0.001                                                      ene-23-yne-26,27-                                                             hexafluorocholecalciferol                                                     ______________________________________                                    

Oral dosage forms comprising compounds of formula I of the invention maybe incorporated in capsules, tablets and the like with pharmaceuticallyacceptable carrier materials.

Illustrative of the pharmaceutically acceptable carrier materials whichmay be incorporated into capsules, and the like are the following: abinder such as gum tragacanth, acacia, corn starch, or gelatin; anexcipient such as dicalcium phosphate, a disintegrating agent such ascorn starch, potato starch, algenic acid, and the like; a lubricant suchas magnesium stearate, a sweetening agent such as sucrose, lactose, orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. Various other materials may be present as coating or tootherwise modify the physical form of the dosage unit. For instance,tablets may be coated with shellac, sugar, or both. A syrup or elixirmay contain the active compound, sucrose as a sweetening agent, methyland propyl parabens as preservatives, a dye, and a flavoring such ascherry or orange flavor.

Topical dosage forms comprising compounds of formula I of the inventioninclude: ointments and creams encompassing formulations havingoleaginous, adsorbable, water-soluble and emulsion-type bases such aspetrolatum, lanolin, polyethylene glycols and the like.

Lotions are liquid preparations and vary from simple solutions toaqueous or hydroalcoholic preparations containing finely dividedsubstances. Lotions can contain suspending or dispersing agents, forexample, cellulose derivatives such as ethyl cellulose, methylcellulose, and the like; gelatin or gums, which incorporate the activeingredient in a vehicle made up of water, alcohol, glycerin and thelike.

Gels are semi-solid preparations made by gelling a solution orsuspension of the active ingredient in a carrier vehicle. The vehicles,which can be hydrous or anhydrous, are gelled using a gelling agent,such as, carboxy polymethylene, and neutralized to a proper gelconsistency with the use of alkalies, such as, sodium hydroxide andamines, such as, polyethylenecocoamine.

As used herein, the term "topical" denotes the use of the activeingredient, incorporated in a suitable pharmaceutical carrier, andapplied at the site of the inflammation for the exertion of localaction. Accordingly, the topical compositions include thosepharmaceutical forms in which the compound is applied externally bydirect contact with the skin. The topical dosage forms comprise gels,creams, lotions, ointments, powders, aerosols and other conventionalforms for applying medication to the skin obtained by admixing thecompounds of formula I with known pharmaceutical topical carriermaterials. In addition to application to the skin, the topicalcompositions of this invention can also be employed in the treatment ofinflammations of mucous membranes, where such membranes are accessibleto topical application of medication. For example, the topicalcomposition can be applied to the mucous lining of the mouth or lowercolon.

EXAMPLE 1

[3aS-[3(S*),3[3aα,7α,7aβ]]-1,1,1-Trifluoro-6[3a, 4, 5, 6, 7,7a-hexahydro-3a-methyl-7-[(trimethylsilyl)oxy]-1H-inden-3-yl]-2-(trifluoro-methyl)-3-heptyn-2-ol

In a 50 ml heart-shaped flask fitted with a gas inlet was placed asolution of (1.79 mmole) 522 mg of [3aS-[3(S*), 3aα,7α,7aβ]]-[[3a,4,5,6,7,7a-hexahydro-3a-methyl-3-(1-methyl-3-butynyl)-1H-inden-7-yl]oxy]trimethylsilanein 15 ml of anhydrous tetrahydrofuran. After cooling at -75° C., 1.85 ml(2.96 mmole) of 1.6M solution of n-butyllithium in hexane was addeddropwise over 5 minutes and the mixture was stirred at -75° C. for 30min. Then a stream of hexafluoroacetone was bubbled into the mixture for15 min with temperature maintained at -75° C. The reaction mixture wasstirred for one additional hour, and then quenched with 1:1 mixture of2M KHCO₃ and 1M Rochelle salt added dropwise. The mixture was stired atroom temperature for one hour and then diluted with 25 ml of the samesalt solution. After extraction with 4×100 ml of CH₂ Cl₂, the organicphase was washed with 50 ml of the same salt solution, dried (Na₂ SO₄)and evaporated. The oily residue was azeotroped with benzene to give2.38g of crude oily product. Purification was performed by flashchromatography (ethylacetate (EtOAc) -hexane 1:9) to give 817 mg (100%)of the title compound as an oil; [α] ²⁵ D+41.0 (c 0.20 CHCl₃); ¹ H NMR(CDCl₃): δ0.07 (s, 9H), 1.01 (s, 3H), 1.10(d, J=8Hz, 3H), 2.15-2.50(m,4H), 3.00 (s,1H),4.08(br s, 1H),5.36(br s, 1H).

EXAMPLE 2[3aR-[1(R*),3aα,4β,7aβ]]-3,3a,5,6,7,7a-Hexahydro-7a-methyl-1-[6,6,6-trifluoro-5-hydroxy-1-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-ol

Into a 100 ml flask with a gas inlet tube was placed a solution of 812mg (1.78 mmole) of[3aS-[3(S*),3aα,7a,7aβ]]-1,1,1-trifluoro-6[3a,4,5,6,7,7a-hexahydro-3a-methyl-7-[(trimethylsilyl)oxy]-1H-inden-3-yl]-2-(trifluoromethyl)-3-heptyn-2-olin 18 ml of anhydrous tetrahydrofuran. To this was added 5.34 ml (5.34mmole) of tetra-butylammonium fluoride in tetrahydrofuran, and themixture was stirred at room temperature under argon for 80 min. Thereaction was then quenched by addition of 9 ml of half-saturated NaHCO₃and stirred at room temperature for an additional 20 min. Excess oftetrahydrofuran was removed by evaporation and additional 9 ml ofbicarbonate was added. The mixture was extracted with 3×120 ml ethylacetate, the extract was washed with brine, dried (Na₂ SO₄) andevaporated to give 890 mg of crude product. After purification by flashchromatography (EtOAc-hexane 1:2), it gave 690 mg of the compound, whichwas crystallized from pentane: 546 mg (79.8%), m.p. 109°-111° C. (inaddition of 90 mg of partially crystalline mother liquors); [α]²⁵D+19.3°(c0.29, CHCl₃); ¹ H-NMR(CDCl₃):δ1.07(s,3H),1.11 (d, J=8Hz,3H),2.01 (m,1H), 2.20-2.53 (m,4H),4.19(br s,1H),5.41(br s,1H).

EXAMPLE 3 [3aR-[1(R*),3aα,4β,7aβ]]-3,3a,5,6,7,7a-Hexahydro-7a-methyl-1-[6, 6,6-trifluoro-5-hydroxy-1-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-one

In a 25 ml heart-shaped one-necked flask fitted with a gas inlet tubewas placed a solution of 100 mg (0.26 mmole) of [3aR-[1(R*), 3aα,4β,7aβ]]-3,3a,5,6,7,7a-hexahydro-7a-methyl-1-[6,6,6-trifluoro-5-hydroxy-1-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-olin 6 ml of anydrous CH₂ Cl₂. To this was added at room temperature, 176mg (0.8 mmole) of pyridinium chlorochromate in several portions, and themixture was stirred at room temperature for 50 min under argon. To thismixture was then added 9 ml of ether in portion and stirring in thecourse of 10 min, then filtered and the filtrate evaporated to dryness.117 mg of crude product thus obtained was purified by chromatography onsilicagel column with ethyl acetate-hexane 1:3 to give the titlecompound 73 mg (73.4%) as amorphous solid; [α]²⁵ D +35.3°(c 0.15,CHCl₃);1H-NMR (CDCl₃): δ0.84 (s, 3H), 1.17 (d, J=8 Hz, 3H), 1.70-1.95 (m, 2H),1.86-1.95 (m, 1H), 2.85 (m, 1H), 3.10 (s, 1H), 5.40 (bs 1H).

EXAMPLE 4 1,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol

In a 50 ml egg-shaped flask fitted with a gas inlet was placed asolution of 333 mg (0. 571 mmole) of[3S-(3α,5β,Z)]-2-[2-[2-methylene-3,5-bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in 7 ml anhydrous tetrahydrofuran. To this was added at-75° C., 0.325 ml (0.521 mmole) of 1.6M n-butyllithium in hexanedropwise under argon, when a red color of reaction mixture developed.After stirring for 6 min, a solution of 73 mg (0.191 mmole) of[3aR-[1(R*),3aα,7aβ]]-3,3a,5,6,7,7a-hexahydro-7a-methyl-1-[6,6,6-trifluoro-5-hydroxy-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-onein 5 ml anhydrous tetrahydrofuran was added dropwise over 15 min period.The reaction mixture was stirred for 1 hour in dark at -75° C. and thenquenched with 2.6 ml of 1:1 mixture of 2N Rochelle salt and 2N KHCO₃solutions and was allowed to warm to room temperature. It was thendiluted with 10 ml of the same salt solution and extracted with 3×60 mlethyl acetate. The extract was washed with 3×30 ml brine, dried (Na₂SO₄) and evaporated. The crude intermediate was purified by flashchromatography on silica gel column with ethyl acetate-hexane 1:5 togive 93 mg (65.2%) of the disilyl-protected title compound.

In a 25 ml dark wall heart-shaped flask fitted with a gas inlet wasplaced 92 mg (0.123 mmole) of the disilyl-protected title compound in 5ml anhydrous tetrahydrofuran. To this was added 0.89 ml (0.89 mmole) of1M tetrabutylammonium fluoride in tetrahydrofuran, and the mixture wasstirred for 16 hrs under argon The reaction was then quenched with 3 mlof half-saturated NaHCO₃ and stirred 20 min at room temperature. It wasthen extracted with 3×50 ml of ethyl acetate. The extract was washedwith 2×15 ml of half-saturated NaHCO₃ and 15 ml of brine, dried (Na₂CO₃) and evaporated. The crude product was purified by flashchromatography with ethyl-acetate 4:1, to give 57 mg (89.4%) of thetitle compound as a foamy glass; [α]²⁵ D+59.1° (c 0.11, CH₃ OH); ¹H-NMR(CDCl₃): δ-0.71 (s,3H), 1.14 (d, J-8 Hz,3H), 2.61 (m, 1H), 2.83 (m,1H), 3.20 (brs, 1H), 4.25- (br s, 1H), 4.26(br s, 1H), 5.02 (s, 1H),5.35 (s,1H),5.42 (s, 1H), 6.10 (d, J=12 Hz,1H), 6,38 (d, J=12 Hz, 1H);UVmax (ethanol) 263 nm.

EXAMPLE 5 25-Hydroxy-16-ene-23-yne-26,27-hexafluoro-cholecalciferol

In a 50 ml egg-shaped flask fitted with a gas inlet was placed asolution of 273 mg (0.604 mmole) of[5S-Z]-2-[2-[2-methylene-5-[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl phosphine oxide in 6.4 ml anhydrous tetrahydrofuran. To thiswas added, at -75° C., 0.343 ml (0.548 mmole) of 1.6M n-butyl lithium inhexane dropwise under argon, until red color of the reaction mixturedeveloped. After stirring for 6 min, a solution of 83 mg (0.217 mmole)of [3aR-[1(R*),3aα,7aβ]]-3,3a,5,6,7,7a-hexahydro-7a-hexahydro-7a-methyl-1-6,6,6-trifluoro-5-hydroxy-1-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-onein 6.5 ml of anhydrous tetrahydrofuran was added dropwise over 23 minperiod. This reaction mixture was stirred in dark for 1 hr 15 min, andthen quenched with 2.7 ml of a 1:1 mixture of 2N Rochelle salt and 2MKHCO₃ at -75° C., and allowed to warm up to room temperature. It wasdiluted with 10.5 ml of the same salt mixture and extracted with 3×65 mlethyl acetate. The extract was washed with 3×32 ml of saturated brine,dried (Na₂ SO₄) and evaporated. This crude silyl protected titlecompound was purified by flash chromatography on silica gel with ethylacetate-hexane 1:5 to give 78 mg (58.3%) of the silylated intermediate.

In a 25 ml flask fitted with a gas inlet, was placed a solution of 76 mg(0.123 mole) of the silyl intermediate in 3.5 ml anhydroustetrahydrofuran. To this was added 0.70 ml (0.70 mmole) of 1M solutionof tetrabutyl ammonium fluoride in tetrahydrofuran, and the reactionmixture was stirred at room temperature for 16 hrs. It was then quenchedwith 2.5 ml of half-saturated NaHCO₃ and stirred at room temperature for15 min. The mixture was extracted with 3×35 ml ethyl acetate, and theextract was washed with 2×12 ml of half-saturated NaHCO₃ and 14 mlbrine, dried ((Na₂ SO₄) and evaporated. The crude product was purifiedby flash chromatography with ethyl acetate-hexane 1:1 to give 49 mg(79.3%) of the title compound; [α]²⁵ D+77.3°(cO.15,CH₃ OH)1H-NMR(CDCl₃): δ0.72(s,3H), 1.13(d,J=8 Hz, 3H), 2.57(m,1H), 2.82(m,1H),3.95(br s, 1H), 4.84(s, 1H), 5.07 (s, 1H), 6.12 (d, J=12 Hz, 1H), 6.23(d, J=12 Hz, 1H), UV max (ethanol) 263 nm.

EXAMPLE 61α-fluoro-25-hydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol

In a 50 ml egg-shaped flask fitted with a gas inlet was placed asolution of 159 mg (0.338 mole) of[3S-(3α,5β,Z)]-2-[2-[2-methylene-3-fluoro-5[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in 4.3 ml anhydrous tetrahydrofuran. To this was added,at -75° C., 0.201 ml (0.322 mmole) of 1.6M n-butyllithium in hexanedropwise under argon, until red color of the reaction mixture developed.After stirring for 6 min, a solution of 47 mg (0.12 mmole) of[3aR-[1(R*),3aα,7aβ]]-3,3a,5,6,7,7a-hexahydro-7a-methyl-1-[6,6,6-trifluoro-5-hydroxy-1-methyl-5-tetrafluoromethyl)-3-hexynyl]-4H-inden-4-onein 2.5 ml of anhydrous tetrahydrofuran was added dropwise. This reactionmixture was stirred in dark for 1 hr 15 min, and then quenched with a1:1 mixture of 2N Rochelle salt and 2M KHCO₃ at -75° C. and allowed towarm up to room temperature. It was diluted with 10.5 ml of the samesalt mixture and extracted with ethyl acetate. The extract was washedwith saturated brine, dried (Na₂ SO₄) and evaporated. This crude silylprotected title compound was purified by flash chromatography on silicagel with ethyl acetate-hexane 1:5 to give 27 mg (35%) of the silylatedintermediate.

In a 25 ml flask fitted with a gas inlet, was placed a solution of 27 mg(0.0425 mmole) of the silyl intermediate in 1.8 ml anhydroustetrahydrofuran. To this was added 0.257 ml (0.257 mmole) of 1M solutionof tetrabutyl ammonium fluoride in tetrahydrofuran, and the reactionmixture was stirred at room temperature for 16 hrs. It was then quenchedwith half-saturated NaHCO₃ and stirred at room temperature for 20 min.The mixture was extracted with ethyl acetate, and the extract was washedwith half-saturated NaHCO₃ and brine, dried (Na₂ SO₄) and evaporated.The crude product was purified by flash chromatography with ethylacetate-hexane 1:1.3 to give 19 mg (86%) of the title compound as aglass; [α]²⁵ D+71.7°(c0.12,CH₃ OH) 1H-NMR (CDCl₃): δ0.72 (s, 3H),1.15(d,J=8 Hz, 3H), 2.63(m, 1H), 2.82(m, 1H), 4.23(br s, 1H), 5.12 (s,1H), 5.14 (din, J= 48 Hz, 1H), 5.41 (s, 1H), 6.12 (d, J=12 Hz, 1H), 6.40(d, J=12 Hz, 1H) .

EXAMPLE 7 1,25 -Dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferol

In a 50 ml egg-shaped flask fitted with a gas inlet was placed asolution of 605 mg (1.06 mole) of[3R-(3α,5β,Z)-3,5-bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl phosphine oxide in 7 mlanhydrous tetrahydrofuran. To this was added at -75° C., 0.645 ml (1.05mmole) of 1.6M n-butyllithium in hexane dropwise under argon, when a redcolor of reaction mixture developed. After stirring for 6 min, asolution of 162 mg (0.424 mmole) of[3aR-[1(R*),3aα,7aβ]]-3,3a,5,6,7,7a-hexahydro-7a-methyl-1-[6,6,6-trifluoro-5-hydroxy-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-onein 4.5 ml anhydrous tetrahydrofuran was added dropwise over 15 minperiod. The reaction mixture was stirred for 1 hour in dark at -75° C.,and then quenched with 2.6 ml of 1:1 mixture of 2 N Rochelle salt and 2NKHCO₃ solutions and was allowed to warm to room temperature. It was thendiluted with 10 ml of the same salt solution and extracted with 3×60 mlethyl acetate. The extract was washed with 3×30 ml brine, dried (Na₂SO₄) and evaporated. The crude intermediate was purified by flashchromatography on silica gel column with ethyl acetate-hexane 1:8 togive 168 mg of the disilyl-protected title compound.

In a 25 ml dark wall heart-shaped flask fitted with a gas inlet wasplaced 168 mg (0.229 mmole) of the disilylprotected title compound in4.5 ml anhydrous tetrahydrofuran. To this was added 2.7 ml of 1Mtetrabutylammonium fluoride in tetrahydrofuran, and the mixture wasstirred for 43 hrs under argon. The reaction was then quenched with 5 mlof water and stirred 25 min at room temperature. It was then extractedwith 3×100 ml of ethyl acetate. The extract was washed 4 times withbrine, dried (Na₂ SO₄) and evaporated. The crude product was purified byflash chromatography with ethyl-acetate-hexane 3:1 to give 99 mg oftitle compound as foam: [α]²⁵ D+25° C. (cO.2 ethanol).

EXAMPLE 825-Hydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferol

The title compound can be prepared in a manner analogous to theprocedure described in Example 5 by using [5S-Z]-5-[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl phosphine oxide insteadof [5S-Z]-2-[2-[2-methylene-5-[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl phosphine oxide.

EXAMPLE 91α-Fluoro-25-hydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferol

The title compound can be prepared in a manner analogous to theprocedure described in Example 6 by using[3R-(3α,5β,Z)]-3-fluoro-5[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide instead of[3S-(3(3α,5β,Z)]-2-[2-[2-methylene-3-fluoro-5[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl phosphine oxide.

EXAMPLE 10 Oral Dosage Form Soft Gelatin Capsule

    ______________________________________                                                              mg/Capsule                                              ______________________________________                                        Compound A              0.0001-0.010                                          Butylated Hydroxytoluene (BHT)                                                                        0.016                                                 Butylated Hydroxyanisole (BHA)                                                                        0.016                                                 Fractionated Coconut Oil (Neobee M-5)                                                                 160.0                                                 ______________________________________                                    

1. Suspend the Butylated Hydroxytoluene and Butylated Hydroxyanisole infractionated coconut oil. Warm to about 50° C. and stir until dissolved.

2. Blanket the solution in step 1 with nitrogen and add Compound A. Stiruntil Compound A has dissolved, maintaining the nitrogen blanket.

3. Fill in soft gelatin capsules

Compound A is 25-hydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol.

EXAMPLE 11 Topical Cream

    ______________________________________                                                                mg/gm                                                 ______________________________________                                        Compound A                0.001-1.0                                           Cetyl Alcohol             1.5                                                 Stearyl Alcohol           2.5                                                 Span 60 (Sorbitan monostearate)                                                                         2.0                                                 Arlacel 165 (Glyceryl monostearate                                                                      4.0                                                 and polyoxyethylene glycol stearate blend)                                    Tween 60 (polysorbate 60) 1.0                                                 Mineral Oil               4.0                                                 Propylene Glycol          5.0                                                 Propylparaben             0.05                                                BHA                       0.05                                                Sorbitol Solution         2.0                                                 Edetate Disodium          0.01                                                Methylparaben             0.18                                                Distilled Water q.s. to   100      gm                                         ______________________________________                                    

1. Melt the Cetyl Alcohol, Stearyl Alcohol, Sorbitan Monostearate,Glyceryl Monostearate and Polyoxyethylene Stearate Blend, Polysorbate60, Mineral Oil and a portion (60%) of Propylene Glycol together in astainless steel container at 70° C. in a water bath.

2. Dissolve Butylated Hydroxyanisole and Propylparaben in the materialfrom step 1 and maintain at 70°-72° C. Record the temperature of themelt.

3. Heat the Sorbitol Solution and the water in a suitable container at70°-75° C.

4. Add the Edetate Disodium and Methylparaben to the solutions in step 3and mix until dissolved. Record the temperature of the aqueous phase.

5. Dissolve the appropriate amount of Compound A in another portion(30%) of the Propylene Glycol in a beaker and add this to the materialfrom step 2 while mixing. Rinse the container with the remaining (10%)of the Propylene Glycol and add this to the mixture from step 2.Maintain a nitrogen atmosphere above the product during this andsubsequent steps.

NOTE: Once Compound A is added, steps 5 and 6 must be completed in rapidsuccession.

6. Add the oil phase from step 2 to the aqueous phase from step 5 whileemulsifying with a high shear mixer. Rinse the oil phase container bywithdrawing a portion of the emulsion and add this immediately to therest of the emulsion.

7. Continue mixing and allow the product to cool to 50°-55° C. Remove analiquot for determination of water content and droplet size. Record theresult. Add additional water if necessary.

8. Continue mixing with a paddle mixer until the product cools to roomtemperature. Record the weight of the final product.

9. Transfer the cream to appropriate containers.

NOTE: 1. The manufacturing has to be done in amber light.

2. The final cream should be packaged within 7 days from completion ofits manufacture.

We claim:
 1. A compound of the formula ##STR12## wherein R is hydrogen,hydroxy, or fluorine and X is H₂.
 2. The compound in accordance withclaim 1, wherein R is hydroxy or fluorine.
 3. The compound in accordancewith claim 1, 26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol.
 4. A pharmaceuticalcomposition comprising an effective amount of a compound of the formula##STR13## wherein R is hydrogen, hydroxy or fluoride, X is H₂.
 5. Thecomposition in accordance with claim 4, wherein R is hydroxy orfluorine.
 6. The composition in accordance with claim 4, wherein thecompound of formula I is26,26,26,27,27,27-hexafluoro-1,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol.7. The composition in accordance with claim 4, suitable for oraladministration.
 8. The composition in accordance with claim 4, suitablefor topical administration.